The high incidence of hepatocellular and other types of cancer in iron overload demonstrates that iron plays a key role in the formation of cancer although the mechanisms remain unknown. Reduced expression of E-cadherin and alpha- and Beta-catenin has been correlated with poor prognosis and reduced survival time of patients with hepatocellular carcinoma and the level of E-cadherin expression has been shown to be inversely related to hepatocellular carcinoma histological grade. Independent reports showed that E-cadherin protein was decreased in several hepatocellular carcinoma tissues compared to nontumorous liver tissue. Therefore, both iron overload and loss of expression of paracellular junction protein have been associated with hepatocellular carcinoma but no connection between iron overload and loss of paracellular junction integrity has been established. A second phenomenon that has been associated with numerous cancers including hepatocellular carcinoma is inhibition of apoptosis.Primary hepatocytes in long-term DMSO culture are highly differentiated and an excellent in vitro model for studying growth, gene expression and pathological changes in adult hepatocytes. We have previously demonstrated that primary cultures of rat hepatocytes maintained in DMSO containing media can be loaded with iron using TMH-ferrocene as an iron donor. TMH-ferrocene is an inert molecule but can donate physiologically active iron following metabolism within the cell. Characterization of this system led us to observe two different phenomenon. First, iron decreases paracellular junction integrity. Iron also decreases expression of a series of paracellular junction genes including E-cadherin and Beta-catenin. Second, iron inhibits apoptosis. Because of the high incidence of hepatocellular carcinoma under conditions of iron overload the current research proposal is designed to elucidate the mechanisms of these disparate phenomenon that may be associated with the formation of iron-induced tumors. The Specific Aims of this proposal are: (1) To establish the mechanism by which iron-loading alters function of paracellular junctions; (2) To determine if the iron-mediated effects on paracellular junctions are determined by the level of the labile iron pool. Experimental manipulation of ferritin levels will be used to regulate the level of intracellular labile iron; (3) To determine the mechanism by which iron inhibits apoptosis in hepatocytes: Is iron promoting the function of an anti-apoptotic pathway or inhibiting a pro-apoptotic pathway? We hypothesize that one or more of these processes may play an early role in the formation of hepatocellular carcinoma under conditions of iron-excess and may be necessary for the pathogenesis of the disease process.